GottesmanProject Title Summary We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia coli as a model system. Mutagenesis studies on the ClpA ATPase of the ClpAP protease have identified mutations in conserved residues within the ATPase domains that give dominant negative phenotypes and others that perturb degradation of a ClpXP substrate. Analysis of the biochemical nature of these mutations should provide further insight into how substrates are recognized and processed by this protease. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to present RpoS to the ClpXP protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. In addition to regulation of RpoS degradation, RpoS translation is regulated by DsrA, a small stable 85 nucleotide RNA. A stem-loop at the 5' end of the RNA is necessary for regulation of RpoS, and acts by pairing with the RpoS leader. This disrupts the secondary structure of the RpoS leader, allowing translation. The promoter of dsrA is regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation resides in a minimal promoter region of 36 base pairs, and probably within the region from +1 to 15, suggesting that promoter structures may be mediating temperature regulation. A novel new RNA, RprA, has been identified as a suppressor of dsrA mutants. It also acts to stimulate RpoS translation, although the mechanism of action appears to differ from that for DsrA. Both DsrA and RprA participate in the osmotic shock induction of RpoS. RprA synthesis is regulated by the two component RcsC and RcsB regulators. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein; they are activated by cell surface stress. When they are activated, RpoS synthesis increases in an RprA-dependent fashion. The demonstration of a second small RNA regulator of RpoS, in addition to DsrA, supports the idea that multiple small RNAs may mediate translational regulation in many cases. A collaboration with Dr. Gisela Storz has been started to extrapolate from these and other known small RNAs in E. coli to identify conditions for detecting other novel small RNAs.